Ashwagandha is one of the most valuable medicinal plants, forming the active component or whole of the phytopharmaceutical products for the prevention and cure of a variety of human ailments and diseases. Numerically, Ayurvedic, Unani and Sidha medicinal products based on the plant currently run above one hundred and it is rapidly expanding worldwide. The plants, plant parts, crude extracts and other phytomatrices/preparations of ashwagandha are often sold on the basis of withaferin-A, besides the prepondrance of withanolides on the whole. Increasing evidences suggest that different withanolides are associated with different levels of pharmaco-efficiencies directed towards different ailments. Withaferin-A is one of the most pharmacologically active constituents. The emerging global trends in herbal products not only emphasize on better-descript and specific lable claims but also assert stringencies of compliances of regulatory frameworks like compliances like DSHEA, NHPR and other analogous regulatory acts and statutes. To cater to these and other needs, Therefore, appropriateness of the methodolocal processes for extractions described herein is not only of tremendous importance for the better and improved yields, resource characterization and accurate quantitative estimations existent in planta or in material, but also provides benefits and logistics of isolations, diagnosis, metabolomics etc. The process is valuable and advantageous in global scale of applications and relevance for standardization of ashwgandha botanicals, metabolomic characterization of plants, chemotypes, analyzing the nature and pace of metabolic transitions under physiological, genetical, environmental and biotic perturbations etc.
Withanolides or withasteroids are a group of naturally occurring C28-steroidal lactone molecules of basically triterpene ancestory and are structurally based on an intact or rearranged ergostane framework. The characteristic lactone is formed through conjoining of C22 hydroxyl and C26 carboxylic acid function. Thus, chemically withanolides are defined as 22-hydroxy ergostane-26-oic acid 26,22-lactone. Biogenesis of withasteroids is known to occur highly scarcely like Tacca plantaginea of Taccaceae (Z. L. Chen, B. D. Wang and J. H. Shen, 1988, Phytochemistry 29, 2999), Cassia siamae of Leguminoseae (C. Srivastava, I. R. Siddiqui, J. Singh and H. P. Tewari, 1992, J. Ind. Chem Soc. 69, 111), Ajuga parviflora of Labiatae (P. M. Khan, S. Ahmad, H. Rubnawaz and A. Malik, 1999, Phytochemistry, 51, 669; P. M. Khan, S. Ahmad and H. R. Nawaz, 1999, J. Nat. Prod. 62, 1290; H. R. Nawaz, A. Malik, P. M. Khan and S. Ahmad, 1999, Phytochemistry, 52, 1357) beyond their usual species limited prodigal producers i.e. 16 out of 96 genera of potato family (Solanaceae). These include, besides the well known Ayurvedic, Sidha, and Unani medicinal plant, Ashwagandha (winter cherry, Withania somnifera), Lycium, Datura, Iochroma, Physalis, Tubocapsicum, Solanum etc. Most of these plants are valued and/or used as: herbs or health foods and/or medicinal plants (based on the traditional knowledge of their positive support to body functions and responses), healing and healthful materials (as such or in the form of supplements, nutraceuticals, concoctions, formulations and compositions) althrough individual's age of growing and graying, rejuvenating and as remedial herbal products of acceptance, source of valuable phytoceuticals/phytochemicals with potentials of lead multifunction drugs/nutraceuticals of near future. Withaferin-A (ergostanically named as 4β, 27-dihydroxy-5β, 6β-epoxy-1-oxowitha-2,24-dienolide), the first member of this group of compounds was isolated from the well known Indian medicinal plant Ashwagandha (P. A. Kurup, 1956, Antibiotic principle of the leaves of Withania somnifera, Current Science, India, 25: 57) and its structure elucidated in 1965 (D. Lavie, E. Glotter and Y. Shvo, 1965, Constituents of Withania sonmifera Dun—the structure of withaferin A, J. Chem. Soc. 7517). Besides it is known to occur as one of the major withanolides in other Withania species (W. aristica, W. coagulans, W. futescens), Acnistus arborescens, Iochroma coccinium, Physalis viscosa. Withanolides are known to possess diverse biological and pharmacological activities. Particularly, withaferin-A, has several hard evidences, established through prolific studies of modern and state of the art research approaches of both in vivo and in vitro kind, of having valuable and a range of pharmacological activities including antibacterial (S. Chatterjee and S. K Chakraborty, 1980, Antimicrobial activity of some antineoplastic and other withanolides. Antonie van Leeuwenhoek 46, 59), cytotoxicity of KB cell cultures, inhibition of Sarcoma 180 and Walker intramuscular carcinosarcoma 256 tumors (S. M. Kupchan, W. K. Anderson, P. Bollinger, R. W. Doskotch, R. M. Smith, J. A. S. Renauld, R. H. K. Schnoes, A. L. Burlingame and D. R. Smith, 1969, Tumor inhibitors-active principles of Acnistus arborescens, Isolation and structural and spectral studies of withaferin-A and withacnistin, J. Org. Chem. 34: 3858), retardation of Ehrlich ascites carcinoma, arresting human larynx carcinoma cells at metaphase (B. Shohat, 1973, Antimitotic properties of withaferin-A and Ehrlich ascite tumor cells-cytological observations, Int. J. Cancer 5, 244), immunoactivation (B. Shohat, 1973, Antimittic properties of withaferin-A and Ehrlich ascite tumor cells-cytological observations, Int. J. Cancer 5, 244; M. Ziauddin et. al. 1996, Studies on the immunomodulatory effects of Ashwagandha, J. Ethanopharmacology, 50, 69–76), immunosuppression through inhibition of adjuvant arthritis and the graft versus host reaction (A. Fugner, 1973, Hemmung immunologisch bedingter entzundungen durch das pflanzensteroid withaferin A, Arzneim. Forsch 23, 932), cancer chemoprevention through selective inhibition of cycloxygenase-2 (B. Jayaprakasam and M. G. Nair, 2003, Cyclooxygenase-2 enzyme inhibitory withanolides from Withania somnifera leaves, Tetrahedron 59, 841–849), attenuation of tachyphylaxis to clonidine on electrically stimulated ileum in vitro (H. Matsuda et al., Structures of withanosides I, II, III, IV, V, Vi and VII, new withanolide glycosides, from roots of Indian Withania somnifera Dunal and inhibitory activity for tachyphylaxis to clonidine in isolated guinea-pig ileum, Bioorg. Med. Chem. 9, 1499–1507, 2001). It has also been suggested to be useful in cognitive dysfunctions and cause noortropic effects [J. N. Dhuley, 2001, Noortropic-like effect of Ashwagandha (Withania somnifera) in mice].
One of the most prolific producer plant, Withania (Ashwagandha in Hindi and Sanskrit, winter cherry in English) is the best source of withanolides particularly withaferin-A and has been recognized as a plant with multiple forms of medicine in Ayurveda, the most descript and extensive and one of the major Indian systems of traditional medicines. Currently, besides several forms of pure herbs and herbal extracts of Withania sold all over the world as herbal health helping GRAS (generally regarded as safe) products, Ashwagandha forms active ingredient of more than 100 Ayurvedic medicines, more than 30 Sidha medicines and a good number of Unani medicines.
It is well accepted that non-nutrient secondary metabolites form the basis of pharmacological value of these medicinal herbs. Thus, withanolidal phytochemicals like withaferin-A abundant and characteristic in Withhania and other producer plants are present day molecular medicinals avowal to the clinical significance of the plant and plant parts discerned in traditional medical practices. Thus, high yield isolation of withaferin A for various experimental, industrial and therapeutic utilities is of obvious importance. Furthermore, precision of dose of active ingredient is of fundamental and vital importance in any acceptable present day clinical practice. Accordingly, accurate estimation of its content, upto closest precision of its occurrence in planta or in materia is important to administer qualified dose(s) and ascertain/relate it to quantified effect(s). For these reasons, inter alia, quantitatively near to entirety of extraction are necessitated at both mass scale production and diagnostic analyses. In summation, efficient, effective and economical extraction/recovery of withaferin-A, as a process, is of tremendous importance to industrial, pharmacological, medicultural, analytical and allied clinical and agri-biotechnological and corporate interests.
Thus, this technological process invention draws its importance as relevance to one of the most critical requirement in the relevant withaferin-A based/centred trade and treatments whether as producer plant or plant part biomass or as products themfrom with the core concern of the quantitative isolation of withanolidal component of withaferin-A in the material of test and/or interest.
Although, some general protocols for post-harvest processing of plant materials of Withania species and other plants/plant parts containing withanolides have been adopted and described by a number of researchers interested in the digging out structural forms of the withanolidal molecules existing in the plant(s)/plant parts of interest from time to time. These methods teach most often than not harvesting of the material followed by shade drying of the material to fully de-aqueous state. The methods further teach that the dried materials be powdered and extracted with 100% methanol or 100% ethanol. However, these investigations have not attended the issue of differential extractability of specific withanolides in consonance with the considerations and cognizances of it being imposed by some structural traits, biogenetic and/or inter- and intracellular sequestration(s) etc. Even routine quantitative analyses carried out in the researches of the plant biological or biotechnological nature, have also been as such adopted from the protocols and processes of isolations taught by their chemical researchers counterparts, without assessing them in true analytical sense. To the best of our knowledge, there is no patent on any alternate or more effective or better in correctness or more precise in solvation and recovery of withaferin-A from holding materials nor any physical or biological state(s) etc. Alternative to dried material has ever been researched and requisitioned to invent a process for efficient isolation and accurately monitor quantitative abundance withaferin-A from the relevant plant materials and products therefrom. The methods for isolation of withaferin-A used so far have not been scrutinized to validate their efficiency as strictly defined relative concentrations/recoveries in numerical values. Accordingly, routinely for isolations and estimations of withaferin-A in the test plant or plant part or the tissue type obtained from the field or generated in vitro through culture techniques have been carried out by shade drying, powdering followed by extraction Soxhlet or sonication or microwave extraction in alcohol.
To the best of our knowledge, there is no patent advising a specific way of efficient isolation of withanolidal compounds from plant tissues for yields and/or quantitative diagnostics. In the U.S. Pat No. 6,153,198 Shibnath Ghosal advises that a Withania somnifera glycosides of withanolides (sitoindoisides) could be prepared by extracting root stock with an aqueous-alcoholic solvent (water-methanol or water-ethanol in 1:1 mixture i.e. 50% aqueous solution of alcohol.). The patent advises that this extract can be devoid of whatever withaferin-A that was co-extracted by further partitioning of the preparation with chloroform wherein chloroform preparation be discarded and chloroform insoluble part be processed for making the formulation with low levels of withanolides including withaferin-A.
Thus, the invented extraction process represents a case of exception to the foregoing fact pertaining to the quantitative extraction, recovery and quantitation of withanolides in the plant materials, extracts and preparations therefrom as they are actually present therein. Thus, extraction of withaferin-A in the literature so far has been quantitatively unevaluated to devise an extraction process that facilitates optimal quantitative recoveries and prevent under estimations due to sub-optimal extractions.
Furthermore, as a prior art only air or shade otherwise dried materials have been advised for isolating withaferin-A and then quantitating its amount in such an extract. This invention teaches the process of the fresh using fresh as well as dry plant materials to have full extractions. The use of fresh material avoids the degradative and/or metabolic transformation loss of withaferin-A during the course of such dessiccations. Such dessications may also accompany losses or recalcitrance to efficient extractions that lead to exacerbating errors in estimations and recoveries for quality control in biomass processing, manufacture, product formulation, cultivar evaluation and other diagnostics including those at the pharmacological and pharmacokinetic levels etc.
Besides attending the technical inaccuracies, as above in monitoring and yielding the withaferin-A from the resources when employing the routine methods taught by the published literature and other public domain documents, this invention saves several expenditures in the process for unit amount of withaferin A isolated.
Another concern not addressed by any disclosures in the open literature is the utility of processing fresh herbs for estimations in planta and other advantages associated with the process on account of that. These include better yields, estimations, saving time, space, and costs in drying, alteration in qualitative compositions, contaminations (environmental materials and inclusions particularly growth of microorganisms (molds, bacteria, fungi etc.) and insects and pests) and adverse effects due to them.
Thus, the industrial, economical and pharmacological, analytical and quality control related necessity of the process for economically extracting and estimating full quantitative amounts of withaferin-A from plant materials and products therefrom is quite obvious. This process has been invented in view of the still need for a process and procedure for isolating and purifying withaferin-A from appropriate biomass or material and medicaments in a commercially viable/valuable way for various established and other exploratory uses. Further, it is an analytically essentialilty that directly provides rightful recovery and evaluation of concentration of withaferin-A to from the basis of dose, does regimen and label claims of the products of commercial and pharmacological/clinical significance. Efficient extraction of herbs while preserving their medicinal values is a process art as well as biochemical science. Many extraction companies/groups/individuals might be delivering extracts of sub-optimal potency or compositional description—knowingly or unknowingly either because of lack of their prior experimental evaluation extraction process(s) and other reasons.
While literature describes some methods through which withaferin-A has been advised to be isolated from a plant or plant part [e.g. Dinan L. et al., 2001, ‘Chromatographic procedures for the isolation of plant steroids’ Journal of Chromatography A 935, 105–123, (see pages 118 and 119) advise that withanolides be generally extracted from plant material with methanol or ethanol which should then be depigmented by routine partitioning following mixing with water. The aqueous methanol phase then should be partitioned against chloroform or dichloromethane or diethyl ether to extract out the withanolides. The method does not advise anything like extent of efficiency when applied to specific withanolide including withaferin-A. A. S. Veleiro et al. (2, 3-dihydrojabrosalactone-A, a withanolide from Acnistus breviflorus, Phytochemistry 24, 1799–1802, 1985) isolated some withanolides including withaferin-A from methanolic extract of the dry herb. In another report on radiotracer based biosynthetic studies these workers (biosynthesis of withanolides in Acnistus breviflorus: biogenetic relationships among the main withanolides, Phytochemistry 24, 2573–2575, 1985). Ahmed et al. ‘Withanolides from Physalis peruviana; Phytochemistry 50, 647–651 (1999) have used 95% ethanol as the extractant to visualize the presence of some known and unknown withanolides in the air dried plant material.
G. Roja et al. [‘Tissue cultures of Withania somnifera: Morphogenesis and withanolide synthesis’ Phytotherapy Research 5, 185–187 (1991) have used 100% methanol to extract withanferin A from dried cultured shoots and tissues to quantitate its concentration. Similarly, M. Furmanowa et al. [In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity, Planta Med. 67, 146–149, 2001] have extracted air-dried green parts of the in vitro raised plants in 100% methanol or ethanol for recovery of withanolides including withaferin-A.
Very recently (2003) B. Jayaprakasam and M. G. Nair (Cyclooxygenase-2 enzyme inhibitory withanolides from Withania somnifera leaves, Tetrahedron 59, 841–849) have extracted shade dried and ground leaves of Withania somnifera sequential with dichloromethane-methanol (1:1), 100% methanol and then water to theoretically scope for extraction under three widely discrete polarities to probably qualitatively have as many withanolides as possible including water soluble glyco-derivatives of withanolides. Thus, no specific extractant system has been designed and evaluated for recovery of withaferin-A or other withanolide. No extraction has been made under a system of narrow range polarity available with water-methanol admixture was attempted or evaluated for recovery in a withanolide specific manner. J. Zhao and associates (Withanolide derivatives from roots of Withania somnifera and their neurite outgrowth activities, Chem. Pharm. Bull. 50, 760–765, 2002) have adopted a routine method of isolating and recovering withanolides including withaferin-A from the Withania herb by extracting the root powder with 100% methanol. In the same way, H. Matsuda et al. used 100% methanolic extract of Withania somnifera dried roots to isolate withanolides (including withaferin-A) and withanosides (Structures of withanosides I, II, III, IV, V, Vi and VII, new withanolide glycosides, from roots of Indian Withania somnifera Dunal and inhibitory activity for tachyphylaxis to clonidine in isolated guinea-pig ileum, Bioorg. Med. Chem. 9, 1499–1507, 2001). S. Ray and S. Jha (Production of withaferin-A in shoot cultures of Withania somnifera, Planta Med. 67, 432–436, 2001) have quantitated concentration of withaferin-A in Withania somnifera shoot cultures produced under different compositions of in vitro culture medium after extracting the dried biomass in 100% alcohol (methanol).
Thus, all prior arts of researches for extraction of withaferin-A, none has attended the relative extractability of withaferin-A in different extractant compositions to arrive at the optimal system. For such an analysis, it is prerequisite to have a specific experiment designed and executed to have a range of extractions made with an admixture of water and appropriate alcohol and to have a comparison of recoveries under such conditions using dry and fresh tissue in quantitative terms to invent the most appropriate one for yields and/or estimation of concentrations. No attempts have been made to address this issue and concern.
The focused microwave-assisted extraction experiments of Kaufmann et al. [‘Parameters affecting microwave-assisted extraction of withanolides’ Phytochemical Analysis 12, 327–331 (2001)] on withaferin-A from air dried Iochroma gesnerioides leaves, have assessed six variables viz. nature and volume of extracting solvent, sample moistening before extraction, extraction time, power of micro-wave irradiation and particle size. Their experiments concluded that for microwave extraction most favourable experiments were using powered material (as is usually done even in conventional extractions) previously moistened (600 microlitre per 100 mg dried material) helps improve microwave assisted extraction from the dry material to conceptually advise that the micro-waving in 100% methanol after moistening (as above) air dried leaves, helps in microwave-assisted methanol extraction (thus final extractant being about 95% methanol) of withanolides including withaferin-A because of better wave energy assimilation and promotion of cell disruption by internal superheating facilitating desorption of analytes from the matrix. The comparison has been made with conventional (soxhlet) extractions using 95% ethanol and 5% water. These workers, in another PSE instrumental extraction [Study of factors influencing pressurized solvent extraction, PSE, of polar steroids from plant material. ’ Chromatographia 54, 394–398, 2001; ‘Recent extraction techniques for natural products: microwave-assisted extraction and pressurized solvent extraction’. Phytochemical Analysis 13, 105–113, 2002) that a 1:1 mixture of methanol:water (i.e. 50% methanol) was useful in using pressurized solvent extraction (PSE) technology on air-dried leaf material. However, these revelations are related to operational parameters of the instrumental approaches of withanolide extractions rather than addressing the chemical characteristics/uniqueness of the molecules per se or in conjunction with their properties in biomass. Thus, serious concerns as how to appropriately extract withaferin-A in a solvent specific way as not been addressed nor the putative advantages of processing through the freshly harvested non-desiccated extractions, disjointed from the extraneous parameters of the instrumental mechanization or automation, has factually not been addressed.
Thus, it is clear that concerns like extractions from fresh herbs (for use in a variety of applications like resource authentication, quality assessment, metabolic, metabolomic and pathway analysis systems and devising post harvest technologies, avoiding drying times, space and money, man power and associated safety and contamination risks and hazards etc.) and use of suitably and stringently defined hydrated alcohol extraction with solvent/alcohol percentage narrowly gauged between 50% alcohol: 50% water to 100% alcohol: 0% water has not been worked out with respect to technological, diagnostic, productivity advantages in a withanolide specific way e.g. for withaferin-A. Additionally, it would offer wider ingenuousnesses e.g. in planta or in materia metabolome state for the withanolide. The approaches so devised have tremendous applications to boot accuracy of determinations and improved extractability with add on savings on several points at the bench level as well as at industrial scale in case of scale up strategies/systems, both in instrumentally unaided as well as mechanized/automate operation of the (agro)-chemical technology.